Specialized technologies for epigenetics in plants.
Specialized technologies for epigenetics in plants.
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植物表观遗传学专业技术。
DOI：
10.1007/978-1-62703-011-3_16
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Xiao,Wenyan
中科院分区：
文献类型：
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作者：
Xiao,Wenyan
植物是发现和研究表观遗传现象的极好系统，如转座子沉默、RNAi、印记和DNA甲基化。印迹是指母系或父系等位基因的优先表达，在哺乳动物和植物的生殖发育中起着重要作用。DNA甲基化对于决定印记基因的母系或父系等位基因是表达还是沉默至关重要。在开花植物中，在生殖过程中存在双受精事件：一个精子使卵细胞受精形成胚胎，第二个精子与中央细胞融合产生胚乳。胚乳是植物体内发生印记的组织，MEDEA（MEA）是第一个在胚乳中发现的具有SET结构域Polycomb group的基因，其母本表达受DNA甲基化和去甲基化控制。近年来，在植物印记基因的鉴定和分子机制的研究方面取得了重大进展。到目前为止，大约有350个基因被发现在植物胚乳（拟南芥，玉米和水稻）中具有差异表达。在拟南芥中，许多印迹基因受DNA甲基转移酶1（MET 1）和DNA去甲基化糖基化酶DEMETER（DME）的调控，和/或受Polycomb组蛋白（PRC 2）调控其染色质状态。胚乳中还存在由未知机制调控的母系表达基因。在本实验中，我们详细描述了如何在拟南芥中进行遗传杂交，从种子中分离胚乳组织，确定基因的印迹状态，并通过亚硫酸氢盐测序分析印迹基因的DNA甲基化。
Plants are excellent systems for discovering and studying epigenetic phenomena, such as transposon silencing, RNAi, imprinting, and DNA methylation. Imprinting, referring to preferential expression of maternal or paternal alleles, plays an important role in reproduction development of both mammals and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants.MEDEA(MEA), a SET domain Polycomb group gene, was the first plant gene shown to be imprinted in endosperm, and its maternal expression is controlled by DNA methylation and demethylation. Recently there has been significant progress in identifying imprinted genes as well as understanding molecular mechanisms of imprinting in plants. Up to date, approximately 350 genes were found to have differential parent-of-origin expression in plant endosperm (Arabidopsis, corn, and rice). InArabidopsis, many imprinted genes are regulated by the DNA METHYLTRANSFERASE1 (MET1) and the DNA-demethylating glycosylase DEMETER (DME), and/or their chromatin states regulated by Polycomb group proteins (PRC2). There are also maternally expressed genes regulated by unknown mechanisms in endosperm. In this protocol, we describe in detail how to perform a genetic cross, isolate the endosperm tissue from seed, determine the imprinting status of a gene, and analyze DNA methylation of imprinted genes by bisulfite sequencing inArabidopsis.